Our studies have focused on an analysis of the structure and biosynthesis of glycoconjugates expressing the carbohydrate epitope Galalpha1-3Galbeta1-4GlcNAc. We have established that glycoconjugates bearing this epitope are abundantly expressed n nonprimate mammals and New World monkeys. In contrast, it isnot expressed by Old World monkeys (OWM), apes or man. However, these latter species produce a large quantity of a naturally ocurring antibody, anti-Gal, which has a strict binding specificity for the Galalpha-3Galbeta1-4GlcNAc eiptope on mammalian glycoconjugates. We have determined that the suppression of Galalpha1-3Galbeta1-4GlcNAc epitope expression in OWM results from a diminution of the activity of the enzyme alpha1-3 galactosyltransferase (alpha1-3GT) which catalyzes the following reaction. Galbeta1-4GlcNAc-R + UDP-Gal Galalpha1-3Galbeta1-4GlcNAc-R +UDP Our overall objective is to study this evolutionarily unique enzyme and its biosynthetic products using a combination of molecular biology, enzymology and carbohydrate structural analysis. Cloning studies of the cDNAfor this enzyme have established that the gene for alpha1-3GT has been conserved in Old World primates in a nonexpressed form. Through molecular biology approaches we propose to study several issues concerning this gene including its evolution in mammals, its differential expression in various tissues, the identity of its catalytic domain and the molecular basis for its evolutionary suppression in OWM. It has also been established that there are different patterns of Galalpha1-3Galbeta1-4GlcNAc glycoconjugate expression among various mammalian species. To understand the biosynthetic factors that result in different patterns of expression of glycoconjugates with the Galapha1-3Galbeta1-4GlcNAc epitope, we propose to carry out carbohydrate structural analyses and enzyme acceptor specificity studies. Proton NMR, FAB-MS and antibody immunostaining methods will be used to elucidate carbohydrate structures, and a novel ELISA based glycosyltransferase assay will be used for the enzyme studies. The combination of molecular biology and biochemical approaches will enable the elucidation of the mechanism(s) which results in the differential expression of the Galalpha1-3Galbeta1-4GlcNAc epitope.